In vitro incorporation of GPI-anchored proteins into human erythrocytes and their fate in the membrane.
In many different cells, glycosylphosphatidylinositol (GPI)-anchored molecules are clustered in membrane microdomains that resist extraction by detergents at 4 degrees C. In this report, we identified the presence of such domains in human erythrocytes and examined the ability of exogenously-added GPI-anchored molecules to colocalize with the endogenous GPI-anchored proteins in these detergent-insoluble complexes. We found that the addition to human erythrocytes of three purified GPI-anchored proteins having different GPI lipid moieties resulted in their efficient and correct incorporation into the membrane. The extent of membrane insertion was dependent on the intactness of the GPI lipid moiety. However, unlike the endogenous GPI-anchored proteins, the in vitro incorporated GPI molecules were not resistant to membrane extraction by Triton X-100 at 4 degrees C. In addition, in contrast to the endogenous GPI-anchored proteins, they were not preferentially released from erythrocytes during vesiculation induced by calcium loading of the cells. These results suggest that in vitro incorporated GPI-linked molecules are excluded from pre-existing GPI-enriched membrane areas in human erythrocytes and that these microdomains may represent the sites of membrane vesicle formation.
Civenni
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Institute of Biochemistry and Molecular Biology, the University of Bern, Bern, Switzerland.
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Brodbeck
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Bütikofer
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D000110:Acetylcholinesterase / Q000097:blood; D002118:Calcium / Q000494:pharmacology; D004910:Erythrocyte Membrane / Q000378:metabolism*; D019856:Ethanolamine; D017261:Glycosylphosphatidylinositols / Q000097:blood*; D006801:Humans; D007531:Isoflurophate; D008562:Membrane Glycoproteins / Q000097:blood; D008565:Membrane Proteins / Q000097:blood; D017830:Octoxynol / Q000494:pharmacology; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010739:Phospholipase D / Q000378:metabolism; D012995:Solubility; D014316:Tritium; D010738:Type C Phospholipases / Q000378:metabolism; D014643:Variant Surface Glycoproteins, Trypanosoma / Q000097:blood
D017261:Glycosylphosphatidylinositols; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D014643:Variant Surface Glycoproteins, Trypanosoma; D014316:Tritium; D007531:Isoflurophate; D019856:Ethanolamine; D017830:Octoxynol; D000110:Acetylcholinesterase; D010738:Type C Phospholipases; D010739:Phospholipase D; C065017:glycoprotein phospholipase D; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D002118:Calcium